Optical cavity PCR

ABSTRACT

An LED-driven optical cavity PCR system and method is disclosed for fast, accurate and reliable PCR based diagnostics. An optical cavity comprising two thin light absorbing metal (AU) films is used for uniform light absorption and subsequent photo thermal light-to-heat conversion is employed for PCR thermal cycling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 111(a) continuation of PCTinternational application number PCT/US2016/044255 filed on Jul. 27,2016, incorporated herein by reference in its entirety, which claimspriority to, and the benefit of, U.S. provisional patent applicationSer. No. 62/199,069 filed on Jul. 30, 2015, incorporated herein byreference in its entirety. Priority is claimed to each of the foregoingapplications.

The above-referenced PCT international application was published as PCTInternational Publication No. WO 2017/019768 on Feb. 2, 2017, whichpublication is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

INCORPORATION-BY-REFERENCE OF COMPUTER PROGRAM APPENDIX

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. § 1.14.

BACKGROUND 1. Technical Field

This description pertains generally to nucleic acids amplificationsystems and more particularly to Polymerase Chain Reaction (PCR) thermalcycling systems for medical diagnostic and life science.

2. Background Discussion

There is an urgent need for rapid and accurate diagnostics due to globaloutbreaks of deadly diseases such as Ebola virus disease (EVD), MiddleEast Respiratory Syndrome corona virus (MERS-CoV), and human infectionwith Avian influenza A (H7N9) virus. Polymerase chain reaction (PCR),the “Gold standard” for many medical diagnostic tests, has become animportant technique in the fields of clinical laboratories,environmental science, forensic science and agricultural science.Typically PCR, which requires multiple cycles between 2 or 3 discretetemperatures, takes an hour or more per amplification because of thelarge thermal mass of Peltier based heating blocks as well as slow heattransfer between the heating block and the plastic PCR tube. However,fast/ultrafast PCR is highly desirable for applications such astime-sensitive diagnosis of infectious diseases, methicillin-resistantStaphylococcus aureus (MRSA) and sepsis at the point-of-care (POC) levelbecause fast therapeutic turnaround time (TAT) decreases not only themortality rate but the severe risk for unknowingly transmitting thedisease to others.

Commercial PCR systems using air heating/cooling and capillary tubes ordirect resistive heating can perform 30 thermal cycles within 10 min.However, these systems are generally not suitable for POC testing due totheir high power consumption (800-1000 W maximum) and heavy weight (over20 kg). For POC testing in resource-limited environments, such as indeveloping countries or field laboratories, a fast/ultrafast PCR systemshould be sensitive, selective, portable, robust, simple, easy to useand characterized by low power consumption through miniaturization andintegration.

To accomplish these requirements, micro fluidic approaches forfast/ultrafast PCR systems have been extensively investigated to reduceamplification time by decreasing sample volume (i.e. thermal mass),allowing for fast heat transfer, thus faster thermal cycling with lesspower consumption. The most commonly used method for static microfluidic PCR thermal cycling is resistive heating with micro-fabricatedthin film heaters and a resistance temperature detector (RTD). While thepower consumption is relatively low, this method requires a complicatedfabrication process to integrate the thin film heater and RTD on a chip.

A peltier heat block is also widely used for both static and continuousflow PCR due to the rapid heating and cooling rate, but requires higherpower consumption. For continuous-flow PCR, PCR amplification occurswhen the reaction samples pass thorough discrete temperature zones. Thismethod can produce faster thermal cycling than static PCR, but generallyrequires an external syringe pump for flow control and lacks the abilityto change the cycle number. Another approach includes infrared(IR)-mediated non-contact selective heating of the PCR mixture for PCRthermal cycling using an IR laser or filament lamp, which harnesses thestrong IR absorbance by the water at wavelengths over 1000 nm. However,as the volume of the PCR mixture increases from nanoliter to microliter,the total thermal cycling time also increases from ˜5 min to ˜40 min dueto the limitation of fast heating and cooling of PCR solution.

Accordingly, an object of the present description is a fast/ultrafastPCR system for POC testing that is sensitive, selective, portable,robust, simple, easy to use and characterized by low power consumptionthrough miniaturization and integration.

BRIEF SUMMARY

One aspect of the present description is an optical cavity PCR systemand method driven by a light-emitting diode (LED) for fast, accurate andreliable PCR based diagnostics. An optical cavity comprising twometallic (e.g. Au) thin films configured for uniform light absorptionand subsequent photo thermal light-to-heat conversion is employed forPCR thermal cycling. Simulation results show that temperaturedifferences across the 750 μm-thick cavity are less than 2° C. and 0.2°C. at 94° C. (denaturation) and 68° C. (annealing/extension),respectively. Optical cavity PCR in accordance with the presentdescription shows excellent temperature precision with less than 1° C.temperature variation between cycles and is able to accomplish 30 PCRthermal cycles from 94° C. to 68° C. within 4 min due to low thermalmass and high thermal conductivity of thin Au films. Using the LEDdriven optical cavity PCR method of the present description, nucleicacid (c-MET cDNA) amplification was demonstrated with lowest templateDNA concentration of 10⁻⁸ ng μL⁻¹ (2 copies per μL) within 15 min.

Further aspects of the technology will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing preferred embodiments of the technologywithout placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The technology described herein will be more fully understood byreference to the following drawings which are for illustrative purposesonly:

FIG. 1A shows a perspective view of an optical cavity for nucleic acidamplification through polymerase chain reaction (PCR) with top andmiddle cavity layers partially removed for clarity.

FIG. 1B is a schematic side view of light absorption in the opticalcavity of FIG. 1A.

FIG. 1C shows a schematic diagram of corresponding nucleic acidamplification through polymerase chain reaction (PCR) for the cavity ofFIG. 1A.

FIG. 2A shows a schematic perspective view of the optical cavity PCRdevice of FIG. 1A with an LED light source.

FIG. 2B shows a schematic side view of the LED-driven optical cavity PCRdevice of FIG. 2A.

FIG. 3 shows an expanded side view of the layering of the optical cavityof FIG. 1A.

FIG. 4A through FIG. 4D show images of the calculated temperaturedistribution inside a 750 μm-thick optical PCR chamber for the bottomonly and cavity (top and bottom) heating.

FIG. 5 shows a plot of the temperature profiles of the PCR chambersalong the white arrows in FIG. 4A through FIG. 4D.

FIG. 6 shows a graph of the temperature difference along the z-axis (0μm on x-axis) of the optical PCR chamber of the present description as afunction of PCR chamber height.

FIG. 7 shows the representative temperature profile for 30 PCR thermalcycles from 94° C. to 68° C. using the LED-driven optical cavity fordifferent chamber heights.

FIG. 8A shows the total reaction time of 30 thermal cycles for theoptical cavity PCR device of the present description with varyingchamber heights.

FIG. 8B shows the average rates during 30 PCR cycles and sample standarddeviations for different chamber heights.

FIG. 8C shows the measured temperature distribution at 94° C.(denaturation) and 68° C. (annealing/extension) during 30 thermal cycleswith different chamber height.

FIG. 8D shows the comparison of temperature profiles during thermalcycling between two positions.

FIG. 9A shows a 2% agarose gel image from the bench-top thermal cyclercompared with the optical cavity PCR device of the present description(with a 750 μm-thick PCR chamber) with different cycle numbers from 95°C. to 68° C.

FIG. 9B shows a 2% agarose gel image from the PCR device of the presentdescription with different initial concentrations of template DNA.

FIG. 10 shows a graph summarizing the total reaction time for thebench-top thermal cycler compared with the optical cavity PCR device ofthe present description.

DETAILED DESCRIPTION

The embodiments detailed below are directed to an optical cavity forheating a PCR mixture or like substance (Optical Cavity PCR). In thetypical PCR sensing process, the PCR mixture generally goes through aplurality of heating and cooling cycles to affect the PCR reaction.Thus, rapid and uniform heating of the target sample (e.g. PCR mixture)is highly beneficial to POC testing. While the embodiments detailedbelow are directed to PCR-based sensing, it is appreciated that theoptical cavity of the present description may be incorporated for usewith any process where rapid and uniform heating of a sample is desired.

1. Optical Cavity PCR Configuration

FIG. 1A shows a perspective view of an embodiment of a PCR sensingdevice 10 comprising an optical cavity 20 for nucleic acid amplificationthrough polymerase chain reaction (PCR) with top and cavity layerspartially removed for clarity. FIG. 1B is a schematic side view of theoptical cavity 20, showing light absorption and corresponding nucleicacid amplification through polymerase chain reaction (PCR).

In one embodiment, optical cavity 20 comprises two opposing thin filmsheets or layers (lower thin film 22 a and upper thin film 22 b) thatare spaced apart to define walls of a micro-fluidic thermal cyclingchamber 24, hereinafter referred to as a “PCR chamber” when used for thespecific example of holding a PCR mixture for PCR-based testing. Thinfilms 22 a and 22 b preferably comprise a light absorbing material orare otherwise configured for absorbing light in a manner so as toprovide rapid and uniform heating of the thin films. Both the lower thinfilm 22 a and upper thin film 22 b are deposited on respective lower andupper substrate layers 18, 14, respectively. Optical cavity 20 maycomprise, for example, a single thermal cycling chamber 24, or an arrayof chambers for multiplexed amplification.

In a preferred embodiment, one or more of the thin films 22 a and 22 bcomprise Au. However, other materials or compositions may bealternatively employed, e.g., metals such as silver (Ag), nickel (Ni),titanium (Ti), chromium (Cr), germanium (Ge), palladium (Pd), ruthenium(Ru), tungsten (W), iridium (Ir), platinum (Pt), and any alloys composedof the foregoing metals, or a multi-layer metallic structure composed ofthe foregoing metals or a combination thereof.

Furthermore, the thin film sheets 22 a and 22 b may comprise anon-metallic, light-absorbing material, including graphene, graphite,carbon nanotubes (CNTs), paint, or the like.

In another embodiment, one or more of lower thin film 22 a and upperthin film 22 b may be patterned to increase light absorption byresonance. The patterned thin film can be formed on flat polymericsubstrate 14, 18, and comprise 2-D or 3-D microstructures ornanostructures in the form of one or more of a pillar array, 1D or 2Dgrating, photonic crystal, hemi-sphere, or the like.

A middle cavity layer 16 is disposed in between lower and uppersubstrate layers 18, 14 to define the optical cavity 20 thickness. In apreferred embodiment, lower and upper substrate layers 18, 14 eachcomprise a transparent polymer, such as acrylic glass, e.g., poly methylmethacrylate (PMMA) or like substance that allows transmission of light.

According to a preferred embodiment, the lower and upper substratelayers 18, 14 preferably comprise a transparent or translucentcomposition to allow light to pass through to the optical cavity 20.While the lower and upper substrate layers 18, 14 are detailedthroughout the description as generally comprising PMMA, it isappreciated that such selection of materials is for exemplary purposesonly, and any number of polymeric or translucent/transparent materialsmay be selected for use as a platform for the thin films. The lower andupper substrate layers 18, 14 may also comprise 2D or 3D microstructuresor nanostructures that may comprise one or more of a pillar array, 1D or2D grating, photonic crystal, hemi-sphere, or other patterned or randomstructures (not shown). In one embodiment, the lower and upper substratelayers 18, 14 comprise nanoplasmonic structures or nanoplasmonic afeedback laser cavity on the surface of the wells that are configured tobe illuminated at a resonance wavelength of nanoplasmonic structures andduration that causes plasmonic photothermal heating of the nanoplasmonicstructures.

As seen in FIG. 1A, a pair of ports 25 a, 25 b are in communication tothe optical chamber 20 through the upper substrate layer 14 and middlecavity layer. The ports 25 a, 25 b allow dispensing of the specimen orsample (e.g. PCR mixture). In one embodiment, the PCR mixture isinjected in a first port 25 a such that the PCR mixture fills PCRchamber 24. The second port 25 b allows air to be pushed out of thechamber until the PCR mixture seals the entire chamber and also exitsout second port 25. One or more fluidic valves and/or fluidic controldevices (both not shown) may be employed to facilitate filling the PCRchamber 24.

FIG. 1B shows a schematic of light absorption in optical cavity 20 fornucleic acid amplification through polymerase chain reaction (PCR). Whena light is illuminated through lower substrate layer 18 (e.g., PMMA)with initial intensity I₀, the light is reflected (R₁), absorbed (A₁),and transmitted (T₁) through the lower thin film 22 a. Subsequently, thetransmitted light (T₁) passed through the chamber 24 and is alsoreflected (R₂), absorbed (A₂), and transmitted (T₂) through the upperthin film 22 b. The thin films 22 a and 22 b act as a plasmonic photothermal light-to-heat converter for PCR thermal cycling. The absorbedlight A₁, A₂ contributes to the photo thermal heating of the thin film(e.g., Au) atoms for the thermal cycling of PCR.

FIG. 1C illustrates an embodiment of the PCR process of denaturation,annealing/extension, and copy (amplification) stages that occurs uponheating of the thin films.

In a first step (1), the process includes illuminating the thin films 22a and 22 b with a light for a specified duration, which affects auniform amount of light absorption into the thin films 22 a and 22 b andaccompanying heating of the thin films 22 a and 22 b, thereby raisingthe temperature of a fluid sample (e.g., PCR mixture) in the chamber 24to a selected temperature for a first period to affect denaturationwithin the PCR mixture.

In a second step (2), the thin films 22 a and 22 b are again illuminatedwith the input light for a specified duration, thereby raising thetemperature of a fluid sample (e.g., PCR mixture) in the chamber 24 to aselected temperature for a second period to affect annealing/extensionwithin the PCR mixture.

In a third step (3), the thin films 22 a and 22 b are again illuminatedwith the input light for a specified duration, thereby raising thetemperature of a fluid sample (e.g. PCR mixture) in the chamber 24 to aselected temperature for a third period to affect copying/amplificationwithin the PCR mixture.

In one embodiment, the three steps are repeated for approximately 30 to40 cycles.

Based on this model, the total absorption of the Au film 1 and 2 arethen given by Eq. 1 and Eq. 2:

$\begin{matrix}{{{\sum\; A_{{film}\; 1}} = {I_{0}{A_{1}\left( {1 + \frac{T_{1}R_{2}}{1 - {R_{1}R_{2}}}} \right)}}},} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

$\begin{matrix}{{{\sum A_{{film}\; 2}} = {I_{0}{A_{2}\left( \frac{T_{1}}{1 - {R_{1}R_{2}}} \right)}}},} & {{Eq}.\mspace{14mu} 2}\end{matrix}$where I₀ is the initial intensity of the light from LED, A₁ (and A₂), T₁(and T₂) and R₁ (and R₂) are the absorbance, transmittance andreflectance of the thin film 22 a (and 22 b), respectively.

The thicknesses of the Au films are optimized to have uniform lightabsorption at both thin films (ΣA_(film1)=ΣA_(film2)) for the greatestuniform temperature distribution as well as the greatest total lightabsorption (ΣA_(film1)+ΣA_(film2)). First, the average transmittance,reflectance and absorbance of thin Au films 22 a, 22 b over the emissionwavelength of the LED is calculated from the measured absorption spectraof thin Au films with different thickness and emission spectrum of theLED. (See Table 2). Then, the absorption ratio (ΣA_(film1)/ΣA_(film2))and total absorption (ΣA_(film1)+ΣA_(film2)) are calculated fordifferent combination of top and bottom Au thickness as shown inTable 1. The combination of a 10 nm thickness for the Au thin film 22 a,and a 120 nm thickness for the Au thin film 22 b was found to be optimalfor both the absorption ratio (1.06) and the total absorption of light(70%), which in a preferred configuration may be used as the thicknessesof the thin Au films for the optical cavity 20.

It is also appreciated that alternative materials may be used for tuningthe absorption of the thin films 22 a, 22 b. For example, lower thinfilm 22 a may be comprised of a material that is less absorptive (withmore transmission characteristics) than upper thin film 22 b. Thus,geometry and/or material composition may be used to tune the absorptiveproperties of the thin films 22 a, 22 b.

FIG. 2A shows a schematic perspective view of the optical cavity PCRdevice 10 with an illumination or light source. FIG. 2B shows aschematic side view of the optical cavity PCR device 10. The lightsource is shown as a series of LEDs 34 (e.g., 7 Luxeon Rebel royal blue,LEDS with peak wavelength of 447.5 nm, 6230 mW at 700 mA injectioncurrent) disposed on a platform 32 (e.g., 40 mm round cool base), alongwith a lens 30 for directing input light I₀ toward the optical cavity20. Alternative light sources may include: a laser diode (LD), tungstenlamp, fluorescent lamp, halogen lamp, mercury lamp, xenon lamp, metalhalide lamp, or any combination of the foregoing. It is also appreciatedthat selection of the type of light source, and/or wavelength orintensity of the input light I₀, may have an effect on the resonantfrequency of the thin films 22 a and 22 b, and thus thickness andmaterial selection of the thin films 22 a and 22 b may vary depending onthe nature of the input light I₀.

A reference chamber 26 with type-K thermocouple 28 is placed next to theoptical cavity 20. The reference chamber 26 and optical cavity 20 areconfigured to be covered by the input light beam I₀ waist (e.g., ø=12mm) at the focal length of the lens 30 to ensure both optical cavity 20and reference chamber 26 receive the same intensity of light such thatphoto thermal heating would occur at the same rate. Both optical cavity20 and reference chamber 26 are placed at the focal length of the lens30 (e.g., 25 mm away from the top surface of lens in this configuration)for highest light absorption. In one embodiment, the lens 30 comprises aPolymer Optics 7 Cell Cluster Concentrator Optic array.

It is appreciated that the reference chamber 26 is employed in theconfiguration of FIG. 2A for purposes of providing a reference fortemperature in the chamber. However, in a preferred configuration forPOC testing, the device of the present description may only includeoptical cavity 20, or a plurality or array of multiplexed opticalcavities, without a reference chamber 26. In such case, feedbackregarding the chamber 24 temperature may be obtained via a temperaturesensor (not shown) or other sensing means.

FIG. 3 shows a close up view of the layering of the optical cavity andsubstrate layers. In one embodiment, each of the thin films 22 a and 22b are covered with a passivation layer 23 to prevent PCR reactioninhibition by the metal layer or light absorbing materials. Thepassivation 23 layer may comprise an oxide thin film, a thin polymerlayer, a thin protein layer, or the like. The middle cavity layer 16 hasa height h, and upper and lower PMMA layers 14, 18 have a height, h_(s).In one exemplary embodiment used for testing the apparatus of thepresent description, h_(s)=1.4 mm, L₁=6 mm, L₂=4 mm (for a total cavitylength of 4 mm) and cavity thickness h was varied from 100 μm, 200 μm,400 μm, and 750 μm. While the above dimensions are illustrative of onepotential embodiment, it is appreciated that other configurations arecontemplated.

For the tests described in further detail below, a National Instruments(NI) 9213 16-channel thermocouple module with high speed mode, auto zeroand cold junction compensation (not shown) was used for accuratetemperature acquisition from the type-K thermocouple 28. Temperaturecycling was performed using the LED's 34, an 80 mm cooling fan (notshown), source meter (not shown), and thermocouple 28 all controlledthrough the LabVIEW program.

In one exemplary configuration, 1 mm-thick poly(methyl methacrylate)(PMMA) sheets were used for the top and bottom substrate layers 14, 18of the optical cavity 20 as well as 100, 200, 400, and 750 μm-thick PMMAsheets for the middle cavity layer 16 were cut with a VersaLASER VL-200laser cutting system (Universal Laser System, Inc., Scottsdale, Ariz.,USA). The top substrate layer 14 was incubated in an oven at 56° C. for6 hrs to allow for annealing of damaged regions by laser cutting. Thebottom substrate layer 18 and the middle cavity layer 16 were firstbonded together using thermal bonding performed at 140° F. with apressure of 0.2 metric ton after UV/ozone treatment of the PMMA sheetfor 10 min. Then, the bottom (bonded with cavity chamber layer) and toplayers were cleaned with 70% ethanol twice for 10 min and rinsed withdeionized (DI) water and dried using N₂. Thin Au films 22 a, 22 b withdifferent thicknesses (10, 20, 40, 80, and 120 nm) were deposited on thebottom and top PMMA sheets by electron beam (E-beam) evaporation under abase pressure of 2×10⁻⁷ Torr. A 50 nm-thick SiO₂ passivation layer wasdeposited over the thin Au films 22 a, 22 b by RF sputtering to preventPCR reaction inhibition by the thin Au film. Finally, the bottom (bondedwith cavity chamber layer) and top layers were bonded together to formoptical cavity 20 PCR chamber 24 using thermal bonding performed at 140°F. with a pressure of 0.2 metric ton after UV/ozone treatment of thePMMA sheet for 10 min.

In one embodiment, the optical cavity 20 may be configured for lasing offluorescent emission during the PCR reaction for real-time opticalcavity PCR. Such configuration may provide further enhancement of thesensitivity of real-time cavity PCR compared to conventional real-timePCR.

2. Experimental Results

a. Simulation for the Temperature Uniformity of Optical Cavity PCR.

A set of heat transfer simulations using COMSOL were performed tocharacterize the temperature uniformity inside the optical cavity 20during PCR thermal cycling. FIG. 4A through FIG. 4D show the calculatedtemperature distribution inside a 750 μm-thick PCR chamber for thebottom only and cavity (top and bottom) heating. FIG. 4A and FIG. 4Cshow the calculated temperature distribution for bottom only heating andFIG. 4B and FIG. 4D show the calculated temperature distribution for topand bottom (cavity) heating in the PCR chamber 24 when the temperatureat the center of the chamber (0 μm on x-axis and 375 μm on z-axis)reaches 94° C. for denaturation and 68° C. for annealing/extension,respectively.

FIG. 4A through FIG. 4D provide clear illustration that the opticalcavity (top and bottom) heating of the device of the present descriptionprovides a more uniform temperature distribution compared to bottom onlyheating, especially at 94° C. The temperature gradients across theheight of the chamber (along the white arrow) are plotted in FIG. 5.Cavity heating in accordance with the present description shows bettertemperature uniformity with a difference of only 1.9° C. and 0.2° C.compared to a temperature difference of 14.4° C. and 0.4° C. for bottomonly heating at 94° C. and 68° C., respectively.

Referring to FIG. 6, the effect of PCR chamber height on temperatureuniformity was also investigated. As the height of the PCR chamberdecreases, the temperature difference (ΔT=T_(max)−T_(min)) across theheight of the chamber decreases for both the bottom only and cavityheating configurations, because heat transfer can be more effective forthe smaller volume in a thinner PCR chamber. Furthermore, it isnoteworthy that cavity heating of the device of the present descriptionshows much smaller temperature differences for all chamber heights atboth the denaturation and annealing/extension temperatures. Therefore,employing the cavity heating in accordance with the present descriptionallows for the uniform heating of PCR mixture for efficient and reliablenucleic acids amplification.

b. LED-Driven Optical Cavity PCR Thermal Cycler.

FIG. 7 shows the representative temperature profile for 30 PCR thermalcycles from 94° C. to 68° C. using the LED-driven optical cavity fordifferent chamber heights. The total time of 30 PCR cycles decreases asthe cavity height h decreases, attaining the minimum time (average 235.5sec) with a 100 μm-thick optical cavity 20 PCR chamber 24, and showinggood linearity with an adjusted R₂ value of 0.9899 as shown in FIG. 8A.

Using the thermal cycling result, heating and cooling rates werecalculated. The average rates during 30 PCR cycles and sample standarddeviations were obtained as shown in FIG. 8B. The fastest heating andcooling rates of 7.50±0.46° C. sec⁻¹ and 6.35±0.49° C. sec⁻¹ wereobtained from the 100 μm-thick PCR chamber 24.

FIG. 8C shows the measured temperature distribution at 94° C.(denaturation) and 68° C. (annealing/extension) during 30 thermal cycleswith different chamber height. The maximum and minimum temperaturesattained during each PCR cycle vary within less than 1° C. at 94° C. andless than 0.5° C. at 68° C., showing comparable temperature accuracywith commercially available bench top thermal cyclers. In particular,due to the lower heat mass as well as fast heat transfer between thethin Au film and PCR mixture through a 50 nm-thick SiO₂ layer, theovershoot and undershoot for the cavity PCR are very low (0.85° C. at94° C., 0.25° C. at 68° C.) compared to the bench top thermal cycler(˜2° C. at 94° C. ˜4° C. at 68° C.), when fast cycling is performed,because the bench-top thermal cycler is not designed for fast thermalcycling.

In order to ensure both chambers at position 1 (reference chamber 26)and 2 (optical cavity PCR chamber 24) are heated at the same rate, areference chamber 26 with type-K thermocouple 28 was placed at bothpositions and thermal cycling was performed. FIG. 8D shows thecomparison of temperature profiles during thermal cycling betweenposition 1 and 2, and it is clearly seen that both positions receive thesame intensity of light from the LED for photo thermal heating of thinAu films.

c. Nucleic Acid Amplification Using Optical Cavity PCR.

To verify the LED driven optical cavity PCR system and method of thepresent description, the amplification of nucleic acids (c-MET cDNA,lung cancer biomarker) was demonstrated.

Human HGFR, or c-MET cDNA was used as a template for PCR. Forconventional bench top PCR with recommended concentrations, the PCRreaction consisted of 0.08 μL KAPA2G DNA polymerase, 4 μL 5×KAPA2Gbuffer A, 0.4 μL dNTP mixture, 1 μL each forward and reverse c-METprimers (stock solution 10 μM), 6.7 μL BSA (3% w/v stock solution for afinal concentration of 10 μg μL⁻¹ BSA) and 2 μL template cDNA. Water wasadded to bring the final volume to 20 μL. To increase the amplificationefficiency in the fast cycling cavity PCR, high concentration ofpolymerase and primers were used. The PCR reaction for the cavity PCRconsisted of 0.4 μL KAPA2G DNA polymerase, 2 μL of 5×KAPA2G buffer A,0.2 μL dNTP mixture, 1 μL each forward and reverse c-MET primers (stocksolution 100 μM), 3.3 μL of BSA (3% w/v stock solution for a finalconcentration of 10 μg μL⁻¹ BSA) and 1 μL template DNA. Again, water wasadded until the reaction was brought to a final volume of 10 μL. Theconcentration of c-MET cDNA also varied, and was brought to as low as10-8 ng μL⁻¹ (2 copies per μL).

The PCR mixture was loaded into the optical cavity 20 PCR chamber 24using a pipette and a first port 25 until the second port 25 on theother side of the chamber 24 filled with fluid to ensure no air bubbleswere formed during thermal cycling. The two ports 25 were sealed withPCR sealing tape to ensure no bubble formation or loss of fluid. Theoptical cavity 20 was placed in line with the reference chamber 26 withthe 120 nm-thick Au film on top, as this is the optimal configurationfor uniform light absorption and maximum total absorption of the thin Aufilms. After amplification, a mixture of 10 μL of PCR product (collectedfrom cavity PCR chamber using pipette) and 10 μL of E-Gel sample loadingbuffer (Invitrogen) was loaded onto E-Gel 2% agarose gels with SYBR SafeDNA gel stain (Invitrogen) and run in an E-Gel iBase Power System(Invitrogen) and took gel image with E-Gel Safe Imager Transilluminator.A 50 bp DNA ladder was used to confirm the size of the product. ABio-Rad C1000™ thermal cycler with CFX96 real-time PCR detection systemwas used for the reference PCR system. PCR was performed in 20 μL volumefor the bench top and 5 μL and 10 μL volumes for the cavity PCR withdifferent chamber thickness. In addition to c-MET cDNA, the λ-DNA wasalso used as a template for PCR for initial cavity PCR optimization. ThePCR reaction to amplify a 104-base pair (bp) λ-DNA target with Z-Taq™DNA polymerase included 0.5 μL Z-Taq DNA polymerase, 5 μL of 10×Z-TaqBuffer, 4 μL of dNTP mixture, 4.5 μL of 10 μM primers (each), and 10 μLof bovine serum albumin (BSA) (50 μg) and was brought to 50 μL withPCR-grade water. The final concentration of the template λ-DNA variedfrom 0.01 ng μL⁻¹ to 10 ng uL⁻¹.

FIG. 9A shows a 2% agarose gel image from the bench-top thermal cycler(Bio-Rad C1000™ thermal cycler with CFX96 real-time PCR detectionsystem) and cavity PCR (with a 750 μm-thick PCR chamber 24) withdifferent cycle numbers from 95° C. to 68° C. (Point 1: bench top 10⁻⁴ng μL⁻¹ and 30 cycles, Point 2 bench top NTC Point 3: cavity at 20cycles, Point 4: cavity at 30 cycles, Point 5: cavity at 40 cycles,Point 5: cavity at NTC).

For the bench-top PCR, a 3-step thermal cycling protocol was used. FIG.9A shows a clear trend that as cycle number increases the intensity ofthe band increases for the optical cavity 20.

FIG. 9B shows the 2% agarose gel image from the cavity PCR product withdifferent initial concentrations of template DNA (Point 1 at 10⁻⁵ ngμL⁻¹, Point 2 at 10⁻⁷ ng μL⁻¹, Point 3 at 10⁻⁷ ng μL⁻¹, Point 4 at 10⁻⁸ng μL⁻¹, Point 5 at NTC). There is a clear trend in band intensity asthe concentration changes. Furthermore, 40 cycles of cavity PCR was ableto amplify as low as 10⁻⁸ ng μL⁻¹ (2 copies per μL⁻¹) within 15 min.

The total reaction time for the bench-top and cavity PCR is summarizedin FIGS. 10. A 70% to 80% reduction in total reaction time can beobtained by using cavity PCR in accordance with the present description,although the total reaction times for the KAPA2G Fast PCR kit arealready 20-70% shorter than conventional PCR assays.

In addition to the fast and sensitive nucleic acid amplification, theoptical cavity 20 is highly repeatable and reproducible. Thedemonstration of a robust LED driven optical cavity PCR makes the systemand method of the present description an ideal candidate forimplementation into POC platforms that require fast, accurate andreliable nucleic acids amplification.

3. Summary

The optical cavity PCR device 10 of the present description waseffective not only in fast PCR thermal cycling, but also in reliablenucleic acid amplification comparable to the conventional bench top PCRsystem. Providing the test result within 30 min is highly desirable tomake a single visit viable. The optical cavity PCR device 10 of thepresent description can meet this requirement because the device canaccomplish 30 PCR thermal cycles within 4 to 10 min and amplify nucleicacid concentrations as low as 10⁻⁸ ng μL⁻¹ (2 copies per μL) within 15min.

By optimizing the thickness of thin Au films of the optical cavity 20,light absorption can be uniformly absorbed at the top and bottom thin Aulayers 22 a, 22 b of the optical cavity 20, resulting in excellenttemperature uniformity with a difference of only 1.9° C. and 0.2° C. at94° C. and 68° C., respectively. As a result, the optical cavity PCRdevice 10 of the present description shows excellent repeatability andreproducibility due to the excellent temperature uniformity as well asprecise temperature accuracy. Generally, the faster thermal cycling isdriven, the greater the variation of the temperature across the PCRsample due to thermal inertia. However, in optical cavity PCR, there isnot a significantly large difference in the temperature accuracy withdifferent sample volume ranging from 1.3 μL to 10 μL. This could beattributed to not only the low thermal mass, but also the fast heattransfer between the thin Au film and PCR mixture through the ultrathin50 nm-thick SiO₂ passivation layer.

The power consumption of the tested device was relatively high (˜20 W),because 7 LED were used on a single PCB to have wide beam waist forheating the reference and cavity PCR chambers at a same rate. However,by using two 3 W LEDs for each of the reference chamber 26 and opticalcavity 20 (or individual optical cavities 20 in a multiplexedconfiguration), the power consumption may be further reduced (˜6 W). Theembodiments of the present description are focused on quantitativereal-time PCR using fluorescent detection as well as integratingmultiple PCR wells and multiple LEDs to allow for high throughputmultiplexed amplification.

In conclusion, a novel ultrafast PCR by a LED-driven optical cavity PCRthermal cycler was demonstrated. The thin Au films of differentthicknesses on both the top and bottom of the cavity shows increasedlight-to-heat conversion efficiency and improved temperature uniformitythan bottom only heating photonic PCR. When controlled for totalamplification time, comparable nucleic acids amplification of the c-METgene with commercial bench top thermal cycler was demonstrated.Ultrafast amplification of c-MET gene, thermal cycling between 94° C.(denaturation) and 68° C. (annealing/extension) was achieved within 4-10minutes for 30 cycles with ultrafast heating. In addition, wedemonstrated repeatability and reproducibility of our cavity PCRplatform. We propose that this simple and robust ultrafast cavity PCRthermal cycler is suitable for POC diagnostics.

From the description herein, it will be appreciated that that thepresent disclosure encompasses multiple embodiments which include, butare not limited to, the following:

1. An apparatus for thermal cycling of a fluidic sample, the apparatuscomprising: at least one micro-fluidic thermal cycling chamber, saidchamber defined by a plurality of chamber walls configured to hold thefluidic sample; a first thin film disposed on a first substrate todefine a first chamber wall of the thermal cycling chamber; a secondthin metal film disposed on a second substrate to form a second chamberwall opposite the first chamber wall; and a light source configured toilluminate the first thin film; wherein a first portion of lightilluminated onto the first thin film is absorbed into the first thinfilm and a second portion of the light illuminated onto the first thinfilm is transmitted through the first thin film; wherein the lighttransmitted through the first thin film illuminates the second thinfilm; wherein at least a portion of the transmitted light illuminatedonto the second thin film is absorbed into the second thin film; andwherein the absorbed light into the first thin film and second thin filmare configured to elevate the temperature of the first thin film andsecond thin film to heat the fluidic sample within the thermal cyclingchamber.

2. The apparatus of any preceding embodiment, wherein one or more of thefirst thin film and second thin film comprise a metallic layer.

3. The apparatus of any preceding embodiment, wherein the metallic layercomprises a metal selected from the group consisting of: gold (Au),silver (Ag), nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge),palladium (Pd), ruthenium (Ru), tungsten (W), iridium (Ir), or platinum(Pt).

4. The apparatus of any preceding embodiment: wherein one or more of thefirst thin film and second thin film comprise a multi-layer metallicstructure; and wherein the metallic structure comprises one or moremetals selected from the group consisting of: gold (Au), silver (Ag),nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge), palladium(Pd), ruthenium (Ru), tungsten (W), iridium (Ir), or platinum (Pt).

5. The apparatus of any preceding embodiment, wherein one or more of thefirst thin film and second thin film comprise a non-metallic lightabsorbing material selected from the group consisting of: graphene,graphite, carbon nanotubes (CNTs), or paint.

6. The apparatus of any preceding embodiment, wherein one or more of thefirst thin film and second thin film comprise a patterned surface toincrease light absorption by resonance.

7. The apparatus of any preceding embodiment, wherein one or more of thefirst substrate and second substrate comprise a translucent materialconfigured to allow transmission of the illuminated light through atleast the first substrate to the first thin film.

8. The apparatus of any preceding embodiment, wherein one or more of thefirst substrate and second substrate comprise 2-D or 3-D microstructuresor nanostructures in the form of one or more of a pillar array, 1D or 2Dgrating, photonic crystal, or hem i-sphere.

9. The apparatus of any preceding embodiment, wherein the first thinfilm has a first thickness, and the second thin film has a secondthickness different than the first thickness.

10. The apparatus of any preceding embodiment, wherein the first thinfilm thickness and second thin film thickness are selected so as tomatch a rate of absorption of light into the first thin film and secondthin film such that the first thin film and second thin film have asubstantially uniform rate of temperature elevation.

11. The apparatus of any preceding embodiment, further comprising atleast one temperature sensor configured to sense the temperature withinthe thermal cycling chamber.

12. The apparatus of any preceding embodiment, wherein the first thinfilm and second thin film have a surface covered with passivation layerto prevent PCR reaction inhibition within the thermal cycling chamber.

13. The apparatus of any preceding embodiment, wherein the light sourceis selected from the group consisting of: a light-emitting diode (LED),laser diode (LD), tungsten lamp, fluorescent lamp, halogen lamp, mercurylamp, xenon lamp, metal halide lamp, or combination thereof.

14. The apparatus of any preceding embodiment, further comprising: firstand second ports coupled to the thermal cycling chamber; wherein thefirst and second ports are configured to allow input of the fluidicsample into the thermal cycling chamber.

15. A method for performing ultra fast thermal cycling of a fluidicsample, the method comprising: providing a micro fluidic thermal cyclingchamber defined by opposing first and second thin films; filling thethermal cycling chamber with the fluidic sample; illuminating the firstthin film with a light source; wherein a first portion of lightilluminated onto the first thin film is absorbed into the first thinfilm and a second portion of the light illuminated onto the first thinfilm is transmitted through the first thin film; illuminating the secondthin film with the light transmitted through the first thin film;wherein at least a portion of the transmitted light illuminating thesecond thin film is absorbed into the second thin film; uniformlyelevating the temperature of the first thin film and second thin film asa function of the absorbed light into the first thin film and secondthin film; and heating the fluidic sample within the thermal cyclingchamber as a result of the elevated temperature of the first thin filmand second thin film.

16. The method of any preceding embodiment, wherein illumination of thefirst thin film is intermittently applied to perform ultra fastmicro-fluidic polymerase chain reaction (PCR) of the fluidic sample.

17. The method of any preceding embodiment, wherein uniformly elevatingthe temperature of the first thin film and second thin film comprises:illuminating the first and second films for a first duration to raisethe temperature of the fluid sample in the thermal cycling chamber to aselected temperature for a first period; illuminating the first andsecond films for a second duration to raise the temperature of the fluidsample in the thermal cycling chamber to a selected temperature for asecond period; illuminating the first and second films for a thirdduration to raise the temperature of the fluid sample in the thermalcycling chamber to a selected temperature for a third period; andrepeating a cycle of illumination periods for multiple cycles to amplifythe fluid sample.

18. The method of any preceding embodiment, wherein the first thin filmhas a first thickness, and the second thin film has a second thicknessdifferent than the first thickness.

19. The method of any preceding embodiment, wherein the first thin filmthickness and second thin film thickness are selected so as to match arate of absorption of light into the first thin film and second thinfilm such that the first thin film and second thin film have asubstantially uniform rate of temperature elevation.

20. The method of any preceding embodiment, further comprising:measuring a temperature within the thermal cycling chamber.

21. The method of any preceding embodiment, wherein the first thin filmand second thin film have a surface covered with a passivation layer toprevent PCR reaction inhibition within the thermal cycling chamber.

22. The method of any preceding embodiment, wherein filling the thermalcycling chamber with the fluidic sample comprises: injecting fluidicsample into the cycling chamber through a first port coupled to thethermal cycling chamber; wherein the injected fluid sample pushes airout of a second port coupled to the thermal cycling chamber.

23. The method of any preceding embodiment wherein the optical cavity isconfigured for lasing of fluorescent emission during the PCR reaction.

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

TABLE 1 Optimization of Au film in optical cavity PCR chamber Ratio/ AuFilm (nm) Total absorption 10 20 40 80 120 Au 10 2.10/0.48 1.64/0.561.32/0.63 1.06/0.69 1.04/0.70 Film 20 4.44/0.51 3.36/0.56 2.64/0.612.14/0.65 2.09/0.65 (nm) 40 15.34/0.54  11.21/0.56  8.58/0.58 7.00/0.596.84/0.60 80 174.78/0.62  126.47/0.62  96.13/0.62  78.57/0.63 76.77/0.63  120 1970.97/0.63   1425.4/0.63  1082.9/0.63  885.19/0.63 864.94/0.63 

TABLE 2 Averaged transmittance, reflectance and Absorptance of the thinAu film over emission wavelength of LEDs with different thickness Authickness T (%) R (%) A (%) 10 nm 50.6 19.7 29.7 20 nm 29.6 31.2 39.2 40nm 10.6 39.6 49.8 80 nm 1.1 37.4 61.5 120 nm  0.1 36.8 63.1

What is claimed is:
 1. A method for performing ultra fast thermalcycling of a fluidic sample, the method comprising: providing amicro-fluidic thermal cycling chamber defined by opposing first andsecond thin films; filling the micro-fluidic thermal cycling chamberwith the fluidic sample; illuminating the first thin film with a lightsource; wherein a first portion of light illuminated onto the first thinfilm is absorbed into the first thin film and a second portion of thelight illuminated onto the first thin film is transmitted through thefirst thin film; illuminating the second thin film with the lighttransmitted through the first thin film; wherein at least a portion ofthe transmitted light illuminating the second thin film is absorbed intothe second thin film; uniformly elevating a temperature of the firstthin film and a temperature of the second thin film as a function of theabsorbed light into the first thin film and second thin film; andheating the fluidic sample within the micro-fluidic thermal cyclingchamber as a result of the elevated temperature of the first thin filmand second thin film.
 2. The method of claim 1, wherein illumination ofthe first thin film is intermittently applied to perform ultra fastmicro-fluidic polymerase chain reaction (PCR) of the fluidic sample. 3.The method of claim 2, wherein uniformly elevating the temperature ofthe first thin film and second thin film comprises: illuminating thefirst and second thin films for a first duration to raise thetemperature of the fluid sample in the micro-fluidic thermal cyclingchamber to a selected temperature for a first period; illuminating thefirst and second thin films for a second duration to raise thetemperature of the fluid sample in the micro-fluidic thermal cyclingchamber to a selected temperature for a second period; illuminating thefirst and second thin films for a third duration to raise thetemperature of the fluid sample in the micro-fluidic thermal cyclingchamber to a selected temperature for a third period; and repeating acycle of illumination periods for multiple cycles to amplify the fluidsample.
 4. The method of claim 2, wherein the first thin film has afirst thickness, and the second thin film has a second thicknessdifferent than the first thickness.
 5. The method of claim 4, whereinthe first thin film thickness and second thin film thickness areselected so as to match a rate of absorption of light into the firstthin film and second thin film such that the first thin film and secondthin film have a substantially uniform rate of temperature elevation. 6.The method of claim 2, further comprising: measuring a temperaturewithin the micro-fluidic thermal cycling chamber.
 7. The method of claim2, wherein the first thin film and second thin film have a surfacecovered with a passivation layer to prevent PCR reaction inhibitionwithin the micro-fluidic thermal cycling chamber.
 8. The method of claim2, wherein filling the micro-fluidic thermal cycling chamber with thefluidic sample comprises: injecting fluidic sample into the cyclingchamber through a first port coupled to the micro-fluidic thermalcycling chamber; wherein the injected fluid sample pushes air out of asecond port coupled to the micro-fluidic thermal cycling chamber.
 9. Themethod of claim 2, wherein the micro-fluidic thermal cycling chamber isconfigured for lasing of fluorescent emission during the PCR reaction.